CRISISS: A Novel, Transcriptionally and Post-Translationally Inducible CRISPR/Cas9-Based Cellular Suicide Switch

• Published in: International journal of molecular sciences Vol. 24; no. 12; p. 9799
• Main Authors: Amberger, Maximilian, Grueso, Esther, Ivics, Zoltán
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 01.06.2023; MDPI
• Subjects: Alu retrotransposon; Animals; Animals, Genetically Modified; Apoptosis; Cell cycle; Cell death; Cell proliferation; Cell therapy; CRISPR; CRISPR-Cas Systems - genetics; CRISPR/Cas9; Deoxyribonucleic acid; DNA; DNA damage; DNA repair; Gene Editing - methods; Gene expression; gene therapy; Genes; Genetic engineering; Genetic modification; Genetic transcription; Genetically modified organisms; Genomes; Genomics; Humans; Kinases; Lymphocytes; Medical equipment and supplies industry; Medical test kit industry; Neomycin; Nuclease; Peptides; Post-translation; Regenerative medicine; RNA; Sleeping Beauty transposon; Suicide; suicide gene; suicide switch; Tetracycline; Tetracyclines; Transposition; Transposons; United States; suicide switch; Alu retrotransposon; gene therapy; CRISPR/Cas9; Sleeping Beauty transposon; suicide gene
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms24129799

With the ever-increasing developing rate of gene and cellular therapy applications and growing accessibility due to products receiving regulatory approval, the need for effective and reliable safety mechanisms to prevent or eliminate potentially fatal side effects is of the utmost importance. In this study, we present the CRISPR-induced suicide switch (CRISISS) as a tool to eliminate genetically modified cells in an inducible and highly efficient manner by targeting Cas9 to highly repetitive retrotransposons in the human genome, causing irreparable genomic fragmentation by the Cas9 nuclease and resulting cell death. The suicide switch components, including expression cassettes for a transcriptionally and post-translationally inducible Cas9 and an specific single-guide RNA, were integrated into the genome of target cells via mediated transposition. The resulting transgenic cells did not show signs of any impact on overall fitness when uninduced, as unintended background expression, background DNA damage response and background cell killing were not observed. When induced, however, a strong expression of Cas9, a strong DNA damage response and a rapid halt of cell proliferation coupled with near complete cell death within four days post-induction were seen. With this proof-of-concept study, we present a novel and promising approach for a robust suicide switch with potential utility for gene and cell therapy in the future.